CD8+ T-cell exhaustion during chronic human immunodeficiency virus (HIV) infection is characterized by increasing expression of inhibitory markers on the cell surface that lead to decreased effector function and dampened immune responses that are essential to achieving therapeutic vaccine efficacy. Inhibitory marker PD-1 is upregulated on exhausted CD8+ T-cells, and blockade with a monoclonal antibody (aPD-1) can help reverse exhaustion. We hypothesized that dosing with aPD-1 would boost the immune system and decrease expression of other exhaustion markers throughout infection to improve therapeutic vaccine responses. To investigate this hypothesis, we studied how exhaustion progresses over time in simian-human immunodeficiency virus (SHIV)-infected Rhesus macaques treated with a novel combinatorial therapeutic regimen consisting of a conserved-elements vaccine to circumvent viral mutants, GS986 to reverse latency, CCR5 gene editing to prevent viral entry, and aPD-1 to reverse CD8+ T-cell exhaustion. We characterized exhaustion in peripheral blood mononuclear cells and mesenteric lymph nodes with surface staining and flow cytometry, with a focus on exhaustion markers PD-1, TIGIT, CTLA-4, LAG-3, and TIM-3 at various timepoints throughout infection and vaccination. Although we observed no differences in viral burden between treatment groups, we observed higher CD8+ T-cell vaccine responses in animals receiving aPD-1 compared to vaccinated animals that did not receive aPD-1, suggesting aPD-1 improved vaccine responses. Although we did not find a correlation between PD-1 signaling and any exhaustion markers, we observed a significant negative correlation between CD8+ T-cell vaccine responses and pre-vaccination TIGIT levels (Spearman r= -0.75, p=0.007). We also found that PD-1 and TIGIT are largely independently expressed on CD8+ T-cells. Taken together, these data suggest a role for dual blockade of PD-1 and TIGIT to improve vaccine efficacy in future studies. Defining the impacts of CD8+ T-cell exhaustion on therapeutic vaccine immunogenicity is crucial to improving combinatorial immunotherapy towards a cure for HIV.

Nucleic acid vaccines introduce DNA or mRNA into cells in vivo, instructing them to express antigens from a pathogen resulting in the induction of immune responses that can provide long term protection from that pathogen. They provide many advantages over traditional vaccines including lower cost, improved safety, and the possibility to rapidly update the vaccine since only the genetic sequence of a new variant is required. One drawback of DNA vaccines has been their relatively poor immunogenicity compared to traditional vaccines which has been overcome, to some extent, by using improved delivery methods and co-formulation with plasmids expressing cytokines as adjuvants. Previous studies have established IL-12, as the “gold standard” genetic adjuvant due to its ability to support differentiation of antigen specific CD4+ T cells to produce Th1 cytokines as well as expansion of antigen specific CD8+ T cells to be more cytolytic in vivo. There is growing interest in identifying other adjuvants that not only increase immunogenicity of DNA vaccines but also modulate the types of responses they induce. In this study, we sought to determine if co-administration of an adjuvant cocktail including IL-18, a pro-inflammatory cytokine, and IRF7, a transcriptional activator of type I interferons, along with IL-12 would enhance antibody responses to DNA vaccines expressing SIV and Influenza antigens in a preclinical nonhuman primate model. Plasma samples were collected at different times post vaccination and the effect of the adjuvants on immunogenicity was measured via IgG ELISA and analyzed. After 2 vaccinations, we observed a significant increase (P=0.0272) in antibody responses against SIV gp130 in the adjuvant cocktail group compared to the IL-12 group. These results indicate that combining adjuvants could provide further improvement in DNA vaccine immunogenicity. Additional studies to determine the impact of this adjuvant cocktail on T cell responses are in progress.